3.2.1. DNA extraction using CTAB

This protocol is for the
extraction of DNA from bee abdomens and/or the thorax, using a lysis buffer
containing CTAB, a compound that is able to separate polysaccharides from other
cell materials. The choice of tissues avoids eye contaminants such as pigments,
which can inhibit PCR and other downstream applications. The method can be scaled down for the
extraction of Varroa destructor mites (see the BEEBOOK paper on varroa (Dietemann et al., 2013) for details on sampling) or bee embryos and up for
larger larvae and pupae (see section 1.2. for their collection). Volumes should
be adjusted accordingly based on sample volume (i.e. initial grinding in 5X
sample volume of buffer, ca.25->200 ml). The subsequent two extraction
protocols are simpler, but the CTAB procedure is excellent for problematic
samples and is flexible in terms of tissue disruption, separation, and rescue
of nucleic acids.

Extract only the abdomen and/or thorax if
possible. If a whole animal is
extracted, use a Qiagen or similar column following manufacturer’s protocol for
final purification of extracted DNA in order to reduce pigments that can
inhibit genetic assays.

Add 50µg proteinase K and 25µl of RNase
cocktail.
While this step is optional, proteinase K improves yields by disrupting cell
and organelle boundaries and is critical for extraction of DNA from many
microbes.

Vortex briefly to mix.

Incubate at 55-65°C from several hours to overnight. Invert occasionally during incubation
(e.g., once every 30 minutes for the first two hours).

Centrifuge for 1 min at maximum speed (~14,000 rpm).
Unwanted tissue debris will form a pellet at the bottom of the microcentrifuge
tube.

Transfer liquid to fresh tube, leaving tissue
debris pellet behind.

Add equal volume phenol:chloroform:isoamyl
alcohol (25:24:1).

Invert several times (10-20 times) to mix then
put on ice for 2 min.

Spin at full speed (~14,000 rpms) for 15 min at 4°C.

Transfer upper phase to fresh tube.

Add 500µl cold isopropanol + 50µl 3M NaOAc.

Vortex to mix, then incubate at 4°C > 30 min.
Samples can be stored at ambient temperature at this point for several days if
needed for transport or timing, otherwise 4°C is best.

Spin at full speed (~14,000 rpms) for 30 min at 4°C.

Carefully decant liquid from DNA pellet.

Add 1 ml 4°C 75% EtOH. Tap vortex briefly to loosen pellet.

Spin at full speed for 3 min at 4°C.

Decant liquid from pellet.

Air dry pellet about10 minutes to evaporate all
residual traces of alcohol.
Do not over dry pellet, as it will be hard to resuspend.

Resuspend in 50-100µl nuclease-free water
(overnight at 4°C).

Check DNA quantity and integrity on an agarose
gel.

First, prepare TBE gel buffer (an aqueous
solution with a final working concentration of 45 mM Tris-borate and 1 mM EDTA). This is often prepared
first as a ‘5x’ concentration comprised of 4 g Tris base (FW = 121.14) and 27.5
g boric acid (FW = 61.83) dissolved into approximately 900 mL deionized water.
Add 20 ml of 0.5 M EDTA (pH 8.0) to this solution and adjust the solution to a
final volume of 1l. Confusingly, the ‘working solution’ of this buffer for most
uses is as 0.5x = a 1/10 dilution of the stock buffer.

For a 1.5%
agarose gel on a large-format gel rig, add 3 g of sterile agarose to 200 ml TBE
buffer in a 500 ml or larger Erlenmayer flask, microwave at high heat for ca.
45 s (without boiling). For smaller gel rigs the volume of the gel can be from
50 to 100 ml. Take flask out and swirl vigorously, then heat in the microwave
again until at full boil for 45 seconds, monitoring to avoid spillover. The
agarose must fully dissolve so the liquid is perfectly clear

Let the solution
cool while swirling every minute until the flask can be held for several
seconds without unbearable heat

While hot,
pipette in 10 µl ethidium bromide
solution (EtBr, 0.5 mg/ml, used with caution as EtBr is a carcinogen and
mutagen) and swirl until mixed

Draw the DNA across the gel toward the
anode/positive charge at ca. 100 V depending on the gel rig size and specifications.

Monitor via the blue bromophenol blue stain
movement (which tracks a DNA size fragment of ca. 300 bp in a 1.5% gel),
stopping the gel and visualizing the DNA using ultraviolet light when it has
progressed enough.

DNA can also be quantified via a spectrophotometer
such as the Nanodrop (www.nanodrop.com), following manufacturer’s protocol:
Briefly, after calibration 1 µl of nucleic acid solution is placed onto a
cleaned pedestal, the lid is closed and a reading is taken prior to cleaning by
wiping the pedestal in preparation for the next sample . The machine will
estimate concentration using the equation dsDNA: A260 1.0 = 50
ng/µl.